Deciphering the Function and Regulation of the B Cell Intrinsic a2,6-Sialylation Network

糖基化是一种重要的翻译后修饰，对许多蛋白质的稳定性和功能起着至关重要的作用。在第一个资助期，项目P3的重点是研究2，6-唾液酸残基对小鼠B细胞在稳态和激活过程中发育和功能的影响。为了详细研究这一点，我们建立了B细胞特异性ST6Gal1基因敲除的体内模型，ST6Gal1是一种将末端a2，6唾液酸残基添加到抗体和其他细胞表面受体上的糖结构上的酶。利用这个老鼠模型，可以进行几个有趣的观察。首先，我们能够证明，免疫球蛋白唾液酸化发生在B细胞内，而不是最近提出的免疫球蛋白分泌后。此外，通过对这些小鼠B细胞发育的研究，我们发现这些小鼠的成熟B细胞亚群，包括滤泡B细胞、边缘区B细胞、生发中心B细胞和浆母细胞/浆细胞在脾和腹膜中的数量显著减少。与产生抗体的B细胞亚群的这一缺陷相一致，我们注意到血清免疫球蛋白水平的严重下降。令人惊讶的是，ST6Gal1缺失的强烈的免疫球蛋白亚类特异性效应在稳定状态下变得明显。因此，虽然血清IgG2c几乎不存在，但IgG1、IgG2b和IgG3水平仅受到轻微影响，这表明在稳态期间，2，6-连锁唾液酸残基对免疫球蛋白亚类的转换有影响。然而，当接种T细胞非依赖和T细胞依赖抗原时，可以分别诱导较低但明显可检测到的IgG1/IgG2c或IgG2c抗原特异性抗体反应。这可能表明，通过Toll样受体或其他共刺激分子发出的共刺激信号可能能够克服稳态时IgG2c开关的缺陷。在第二个资助期，我们希望在第一个资助期的结果的基础上，研究B细胞亚群和单细胞水平，决定2，6-连锁蛋白唾液酸化水平的酶在哪里以及何时表达，对于B细胞激活和类别转换至关重要的不同信号通路如何因缺少2，6-连锁唾液酸残基而受到影响，以及在近交系小鼠模型中观察到的表型如何通过产生人类B细胞中ST6Gal1基因被敲除或敲除的人源化小鼠而转化到人类免疫系统中。

Glycosylation is an essential post-translational modification and plays a critical role for the stability and function of many proteins. In the first funding period the focus of project P3 was to study the impact of a2,6 sialic acid residues on mouse B cell development and function during steady state and activation. To study this in detail, we generated an in vivo model for a B cell specific knockout of ST6Gal1, the enzyme that adds terminal a2,6 sialic acid residues to sugar structures present on antibodies and other cell surface receptors. Using this mouse model allowed to make several interesting observations. Firstly, we were able to demonstrate that IgG sialylation occurs within B cells and not post IgG secretion, as suggested more recently. Moreover, by investigating B cell development in these mice, we show that the number of mature B cell subsets including follicular B cells, marginal zone B cells, germinal centre B cells and plasma blasts/plasma cells in the spleen and B2 cells in the peritoneal cavity was strongly reduced. In line with this defect in development of antibody producing B cell subsets, we noted a severe reduction in serum IgG levels. Surprisingly, a strong IgG subclass specific effect of a deletion of ST6Gal1 became evident during the steady-state. Thus, while serum IgG2c was virtually absent, IgG1, IgG2b and IgG3 levels were only slightly affected, suggesting an impact of a2,6-linked sialic acid residues on IgG subclass switching during the steady state. Upon vaccination with T cell-independent and T cell- dependent antigens, however, a reduced but clearly detectable level of IgG1/IgG2c or IgG2c antigen-specific antibody responses could be elicited, respectively. This may suggest, that co-stimulatory signals via toll-like receptors or other co-stimulatory molecules may be able to overcome the defect in IgG2c switching during the steady state. In the second funding period we would like build on the results of the first funding period and study on a B cell subpopulation and single cell level where and when enzymes determining the level of a2,6-linked protein sialyation are expressed, how different signaling pathways critical for B cell activation and class-switching are affected by a lack of a2,6-linked sialic acid residues and how the phenotypes observed in inbred mouse models can be translated to the human immune system via the generation of humanized mice with a knockdown or knockout of ST6Gal1 in human B cells.